In vitro chondrogenic differentiation of human adipose-derived stem cells with silk scaffolds

Abstract

Human adipose-derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with natural and synthetic biomaterials. In the present study, we hypothesized that porous aqueous-derived silk protein scaffolds would be suitable for chondrogenic differentiation of human adipose-derived stem cells. Human adipose-derived stem cells were cultured up to 6 weeks, and cell proliferation and chondrogenic differentiation were investigated and compared with those in conventional micromass culture. Cell proliferation, glycosaminoglycan, and collagen levels in aqueous-derived silk scaffolds were significantly higher than in micromass culture. Transcript levels of SOX9 and type II collagen were also upregulated in the cell-silk constructs at 6 weeks. Histological examination revealed that the pores of the silk scaffolds were filled with cells uniformly distributed. In addition, chondrocyte-specific lacunae formation was evident and distributed in the both groups. The results suggest the biodegradable and biocompatible three-dimensional aqueous-derived silk scaffolds provided an improved environment for chondrogenic differentiation compared to micromass culture.

Keywords: adipose-derived stem cells; cartilage; chondrogenic differentiation; silk scaffolds.

Figure 1.

Proliferation of hASCs. Proliferation of hASCs in the aqueous-derived 3D scaffolds and micromass cultures determined by DNA assay using the PicoGreen assay. Data are shown as mean ± standard deviation from four samples. The symbol “*” represents statistically significant differences (p < 0.05). hASC: human adipose-derived stem cell; 3D: three-dimensional.

Figure 2.

Synthesis of glycosaminoglycans (GAGs). The amount of GAG in samples measured by the 1,9-dimethylmethylene blue (DMB) assay. Data are shown as mean ± standard deviation from four samples. The symbol “*” represents statistically significant differences (p < 0.05).

Figure 3.

Measurement of total collagen. The amount of total collagen measured by the Hride Tullberg–Reinert method. Data are shown as mean ± standard deviation from four samples. The symbol “*” represents statistically significant differences (p < 0.05).

Figure 4.

Expression of RNA transcripts related to chondrogenic differentiation. Transcript expression of several chondrogenic differentiation markers quantified by real-time RT-PCR. a; Aggrecan Gene Expression, b; Type I Collagen Gene Expression, c; Sox 9 Gene Expression, d; Type II Collagen Gene Expression. Transcript levels were normalized by GAPDH. Data are shown as mean ± standard deviation from four samples. The symbol “*” represents statistically significant differences (p < 0.05). RT-PCR: reverse transcription polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Figure 5.

Alcian blue staining. (a and b) Micromass cultures and (c and d) constructs of silk with hASCs cultured for 6 weeks. Extracellular matrices of proteoglycan (blue) were detected in the both groups. Arrow (→) indicates examples of chondrocyte-specific lacunae. Scale bars indicate 250 µm (a and c; 10×) and 100 µm (b and d; 40×).